Oocyte denudation is an important part of routine work of a reproduction laboratory, and involves removing granular cells of an oocyte-corona-cumulus complex which are pre-digested by an enzyme, to assess the maturity of an oocyte and facilitate micromanipulations, such as for intracytoplasmic sperm injection (abbreviated as ICSI), granular cells are required to be removed before cryopreservation of the oocyte; for in vitro fertilization (abbreviated as IVF), such as IVF in microdrop method or IVF in test tube method, it is required to remove granular cells surrounding the oocyte, to assess fertilization condition. The oocyte denudation plays an important role in the above-mentioned manipulations, and directly affects the effect of subsequent manipulation steps.
For decades, oocyte denuding tools remain essentially unchanged, and are completely manually operated. Conventional oocyte denuding pipettes include an oocyte denuding needle and an oocyte denuding Pasteur pipette, which respectively have corresponding latex bulbs sleeved thereon, to constitute respectively a needle type oocyte denuding tool and a Pasteur pipette type oocyte denuding tool. A conventional oocyte denuding method mainly includes repeatedly pressing a latex bulb by fingers to provide a power for suctioning and blowing, to allow an oocyte-corona-cumulus complex pre-digested by hyaluronidase or an oocyte-corona-cumulus complex digested by a sperm after performing an IVF in test tube method to enter and exit from an orifice of an oocyte denuding pipette to remove granular cells, or to allow an oocyte in an oocyte-corona-cumulus complex adhered to the bottom of a culture dish after performing an IVF in microdrop method to be separated from the oocyte-corona-cumulus complex under the action of a suction force, and then to repeatedly blow and suction the oocyte to remove remaining granular cells. In another solution, an oocyte denuding pipette is connected to a mouth pipette by a hose to form a mouth pipette type oocyte denuding tool, and its mouthpiece is placed into an oral cavity, and the oocyte-corona-cumulus complex is by blew and aspirated by the oral cavity repeatedly to enter and exit from an orifice of the oocyte denuding pipette to remove granular cells. According to the tightness of bonding of granular cells of each oocyte-corona-cumulus complex, the degree of difficulty in removal of the granular cells varies, and the number of times of aspirating and blowing ranges from several times to tens of times.
A Patent titled “ORAL BALLOON TYPE OOCYTE DENUDING DEVICE” (Patent No. CN200720157432.9) discloses an oral balloon type oocyte denuding tool for removing an oocyte-corona-cumulus complex around an oocyte. It includes a micropipette and a hose sleeved on a rear end of the micropipette, and a tail end of the hose is connected with a soft balloon. In operation, the balloon is placed between upper teeth and lower teeth of an oral cavity, and is controlled by occluding teeth to provide a power for blowing and suctioning, to allow the oocyte to repeatedly and gently enter and exit from a tip of the micropipette, thereby achieving the purpose of removing most of granular cells surrounding the oocyte. In this invention, the mouthpiece of the mouth pipette is replaced by the balloon, and the effect of suctioning and blowing is achieved by squeezing the balloon through teeth occluding. Compared with the method using the mouth pipette, his method can avoid the risk of polluting the oocyte by expiration.
A Patent “OOCYTE DENUDING NEEDLE” (Patent No.: CN201220219074.0) provides an oocyte denuding needle applicable to oocytes of all sizes. This invention is a hollow oocyte denuding needle for removing granular cells surrounding the oocyte. The invention only makes changes to the structure of the oocyte denuding needle, and using the same oocyte denuding method as the conventional oocyte denuding method.
A Patent “NOVEL TEST-TUBE BABY OOCYTE DENUDING MANIPULATOR” (Patent No. CN201320050412.7) provides an oocyte denuding manipulator consisting of a handheld pressing device and an oocyte suctioning device. The handheld pressing device is composed of a pressing rod cap, a pen cap, a rear shell, a spring, a pressing rod and a front shell, and the oocyte suctioning device is composed of a gasbag, a PS tube, a needle seat and an oocyte denuding pipette. The gasbag, the PS tube, the needle seat and the oocyte denuding pipette are successively installed on a lower-middle portion of the rear shell from top to bottom; and a push button connected to a slide fastener is installed in the middle of the front shell. This invention has a significantly improved manipulation performance compared with the conventional latex bulb.
However, the oocyte denuding apparatuses in the conventional technology have the following deficiencies.
First, the control is inaccurate, and various risks exist. In the conventional oocyte denuding technology, it depends on the muscle power of an operator to control the latex bulb of an elastic operating element, the mouth pipette, the oral balloon, and the gasbag of the oocyte denuding tools. When the latex bulb, the oral balloon and the gasbag are compressed and deformed under the action of an external force, the elasticity possessed by the materials of these members has capabilities of releasing the compressive deformation and restoring to the original state, which is a power source for this kind of manual oocyte denuding tools to generate a suction force. A squeezing force applied to the above elastic operating element by the operator is a power source for this kind of manual oocyte denuding tools to generate a blowing force. For the mouth pipette type oocyte denuding tools, the function of the oral cavity is similar to the function of the elastic operating element of the above oocyte denuding tool, the difference lies in that, in the mouth pipette type oocyte denuding tools, the blowing and the suctioning are both processes in which powers are applied by an operator actively. The processes in which the suctioning force and the blowing force are generated and converted are always accompanied by a counter balance between the pressing force and the restoring force of the elastic element. In oocyte denudation, especially in an initial stage of oocyte denudation, the oocyte-corona-cumulus complex is slightly larger than an inner diameter of an orifice of the oocyte denuding pipette, and may block the orifice of the oocyte denuding pipette when passing through there, which is reflected as a reduction in the velocity of the oocyte-corona-cumulus complex passing through the orifice of the oocyte denuding pipette. After the operator adjusts to increase the suctioning force or the blowing force, especially when increasing the blowing power, once the oocyte-corona-cumulus complex passes through the orifice of the oocyte denuding pipette, the operator cannot stop timely, causing that the balance between the interconnected two processes of suctioning and blowing is lost, and the volume variations caused in the two processes are not consistent with each other, for example, the volume variation caused in the blowing process is greater than the volume variation caused in the suctioning process, resulting in that a series of bubbles are blown out. The oocyte-corona-cumulus complex or the oocyte is adhered to the peripheries of the bubbles, and are floating on the surface of liquid droplets of a culture medium along with the bubbles. The operation interface of oocyte denudation and the bubbles are presented on different focusing planes, and the oocyte-corona-cumulus complex or the oocyte adhered to the bubbles may be located at any position on the surface of the bubbles as well, hence it requires to adjust the focus length of the microscope continuously to look for the oocyte-corona-cumulus complex or the oocyte on the peripheries of the bubbles, causing the total oocyte denuding operation time to be prolonged, which not only increases the risk of damaging the oocyte due to the prolonged in vitro operation time, but also has a substantial risk of losing the oocyte. For trying to avoid generation of the bubbles as much as possible, an operator generally utilizes the oocyte denuding pipette to absorb several microliters of culture medium in advance and remains the absorbed culture medium in the tip of the oocyte denuding pipette, so as to compensate the inconsistent volume variations in the processes of blowing and suctioning, to allow the liquid column within the oocyte denuding pipette to have a height far beyond the height of the liquid in the culture dish.
The inaccurate control in the conventional oocyte denudation method exposes the oocytes or zygotes to a risk caused by temperature fluctuation. Temperature is one of the most important factors affecting the quality of the oocytes, zygotes and embryos. The basal body temperature of a human being is 37 degrees Celsius, the in vitro operation is to try to keep the temperature of the environment where the oocytes or the zygotes are located to be 37 degrees Celsius. Generally, in order to have cleaner air, the oocyte denuding operation is performed on a ventilated ultra-clean bench, the ultra-clean bench has a temperature of about 37 degrees Celsius, a room temperature is about 25 degrees Celsius, and the temperature of the culture medium in the culture dish where the oocyte-corona-cumulus complex is located is close to the temperature of the ultra-clean bench. A tip portion of the oocyte denuding pipette is very thin and small, and in the case that the oocyte is denuded manually, the liquid level of the culture medium in the tip portion of the oocyte denuding pipette is much higher than the liquid level of the culture medium in the culture dish. Only about 0.1 μL of culture medium is in a tip end, submerged in the culture medium in the culture dish, of the oocyte denuding pipette and has a temperature close to the temperature of the culture medium in the culture dish, and a rest part of the oocyte denuding pipette is exposed outside of the culture medium in the culture dish. In this way, the oocyte denuding pipette has two regions with quite different temperatures, which includes a first region, which is the tip end of the oocyte denuding pipette beneath the liquid level of the culture medium in the culture dish, and has a temperature close to that the temperature of the culture medium in the culture dish; and a second region, which is the rest part of the oocyte denuding pipette not submerged in the culture medium of the culture dish and has a temperature close to the room temperature. When the length of the liquid column of the culture medium suctioned into the oocyte denuding pipette is higher than the liquid level of the culture medium in the culture dish, heat is quickly taken away by wind through the glass pipette wall of the oocyte denuding pipette, the oocyte or zygote moves back and forth in the environments with a temperature difference above a few degrees Celsius, causing a risk of damaging the oocyte or zygote due to temperature fluctuations. A low temperature has effects on the oocytes and zygotes including decreasing the level of metabolism and altering organelle morphology and organelle function of the oocytes and zygotes. For example, at a temperature of 33 degrees Celsius, the spindle will begin to be depolymerized within 5 minutes and will completely disappear within 10 minutes. If the same ovum is heated to 37 degrees Celsius, the spindle may be completely recovered within 10 minutes. If the temperature drops to 28 degrees Celsius, the spindle is depolymerized at an increased velocity, and the time period till the spindle completely disappears is shortened, further, it requires a longer time for the spindle to recover after being heated. If the spindle is depolymerized in an environment with a temperature of 25 degrees Celsius, the spindle can hardly recover within twenty minutes after being heated to 37 degrees Celsius. In the recovering period, if cell cycle variations occur, the number of chromosomes will be abnormal. It is generally believed that the temperature of a human oocyte and a zygote during the in vitro manipulation process must be maintained in a relatively constant range, such as 35 degrees Celsius to 37 degrees Celsius, and in the manipulation process, fluctuations of temperature should be minimized as far as possible. In oocyte denudation, for trying to minimize this kind of risk, it requires that the liquid column of the culture medium suctioned into the oocyte denuding pipette should be located below the liquid droplets in the culture dish. As an example that the culture medium in the culture dish where the oocyte-corona-cumulus complex is located has a height about 2.5 mm and an included angle between the oocyte denuding pipette and the bottom of the culture dish is 30 degrees, the length of the liquid column in the oocyte denuding pipette has to be less than 5 mm to make sure that the liquid level of the culture medium in the oocyte denuding pipette is below the liquid level of the liquid droplets in the culture dish. The tip end of the oocyte denuding pipette is very thin and small, and an inner diameter of the tip of the pipette is generally 150 μm, and the volume of the culture medium having a height of 5 mm in the tip of the pipette is about 0.1 μL. In the culture medium with such a tiny volume, it is almost impossible to control the elastic operation element of the oocyte denuding tool through muscle power of the operator to drive the oocyte-corona-cumulus complex to enter and exit from the oocyte denuding pipette under conditions of not generating bubbles and adjusting the velocity appropriately.
Second, the operation has a strong subjectivity, and cannot be standardized. The oocyte denuding operation requires a gentle blow, but the “gentle blow” cannot be explicitly defined, different operators have different subjective feelings on the “gentle blow”, which are reflected as significant differences in the depth and velocity of pressing the latex bulb, pressing the spring pressing rod, expirating/aspirating, and teeth occluding in using the above conventional oocyte denuding tools. These differences are directly reflected as a difference in length of liquid columns of the culture medium suctioned into the oocyte denuding pipette and a difference in velocity of the oocyte-corona-cumulus complexes passing through the orifice of the oocyte denuding pipette, and indirectly reflected as a difference in degree of risks of damaging the oocytes caused by temperature fluctuations and a difference in magnitudes of action forces on the oocyte-corona-cumulus complexes. The flow rate and the pressure of liquid are in a positive relationship, and when the diameter of the spout is certain, the faster the flow rate of the liquid, the higher the pressure at the spout. Controlling the frequency of blowing and suctioning can adjust the flow rate of the liquid, to achieve the adjustment to the pressure at the spout, and further achieve the adjustment to the suctioning force and blowing force applied on the oocyte-corona-cumulus complexes. That is, as the frequency of blowing and suctioning is increased, the suctioning force and the blowing force acted on the oocyte-corona-cumulus complexes are also increased, and further, the acting force for removing the granular cells is also correspondingly increased, and the velocity of the removal is increased. Therefore, the velocity of oocyte denudation may be adjusted by adjusting the frequency of blowing and suctioning. In the conventional oocyte denuding tools, the suctioning force and the blowing force acted on the oocyte-corona-cumulus complexes are adjusted by adjusting the depth and velocity of pressing the latex bulb, pressing the spring pressing rod, expirating/aspirating, and teeth occluding. In one complete oocyte denuding process of the oocyte-corona-cumulus complex, blowing and suctioning may be performed at multiple velocities, and correspondingly there are multiple blowing forces and suctioning forces of different magnitudes. Different blowing forces and suctioning forces and the durations thereof, especially an excessive fierce blowing force and an excessive fierce suctioning force and the durations thereof, may have different degrees of potential negative effects on the oocytes, and if the blowing and suctioning are excessively fierce, the oocyte may be ruptured and disintegrated. In manual oocyte denudation, an operator makes judgment and adjustment completely depending on subjective experiences, and cannot quantify the specific operation details, and correspondingly, cannot provide specific objective data of the whole oocyte denuding process. Whatever the embryos have a high quality or a low quality, when trying analyzing reasons in the oocyte denuding process, it cannot derive a valuable conclusion by reviewing the oocyte denuding operation process or comparing operations performed by different operators.
Third, the working load is huge. In the conventional oocyte denuding process, each time of blowing and suctioning the oocyte-corona-cumulus complex must corresponds to one time of pressing/releasing the elastic element or one action of blowing/aspirating by the oral cavity. In the conventional oocyte denuding tools either operated by a single hand or double hands, or controlled by the oral cavity, for achieving a best control effect, the operator needs to maintain the counter balance always accompanied in the conversion process of the suctioning force and the blowing force, thus, muscles of his fingers or oral cavity of the operator are always in a tensioned state, which results in physical fatigue of the operator. Further, the conventional oocyte denuding tool has risks of damaging the oocytes due to prolonged operation time and temperature fluctuations, and a heavy risk of losing the oocytes, which also places an increased psychological burden on the operator.
Therefore, to address the above technical issues, it is indeed necessary to provide an advanced electric oocyte denuding device and an oocyte denuding method thereof, to overcome the above mentioned drawbacks in the conventional technology.